Bakuchiol Dimers From Psoraleae Fructus That Inhibit Nitric Oxide Production in RAW264.7 Macrophages Cells


 Background: Dried fruit of Psoralea corylifolia L. (Psoraleae Fructus) is one of the most popular traditional Chinese medicine with treatment for nephritis, spermatorrhea, pollakiuria, asthma, and various inflammatory diseases. Bakuchiol is main meroterpenoid with bioactive diversity from Psoraleae Fructus.Methods: Various column chromatography methods were used for isolation experiment. Structures and configurations of these compounds were determined by spectroscopic methods and single-crystal X-ray diﬀraction. Their inhibition on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were evaluated by the Griess reaction.Results: Twelve unpresented bakuchiol dimmers, bisbakuchiols M–U (1–9) and bisbakuchiol ethers A–C (10–12), along with five known compounds (13–17), were isolated from the fruits of Psoralea corylifolia L. Compounds 1–3 and 10–14 exhibited inhibitory activities against LPS-induced NO production in RAW264.7 macrophages, and the inhibition of compound 1 (IC50 = 11.47 ± 1.57 μM) was equal to that of L-NIL (IC50 = 10.29 ± 1.10 μM) as a positive control.Conclusions: Seventeen bakuchiol dimers (1–17), including 12 undescribed dimers and 5 known compounds, were isolated. Bisbakuchiol M (1), whose other bakuchiol unit was cyclized to form a 6/6/5 tricyclic system, was a new skeleton compound. Some compounds exhibited NO inhibition activities and the inhibition of compound 1 was equal to that of L-NIL, a positive control. These findings suggested that Psoraleae Fructus provided natural anti-inflammatory constituents and bisbakuchiol M had the potential to be novel NO inhibitor.


Background
The higher plant, Psoralea corylifolia L. (Cullen corylifolia (L) Me k) is an annual herb and belongs to family Leguminosae distributed in China, India, Malay peninsula, and Indonesia [1]. Dried fruit of P. corylifolia (Psoraleae Fructus) is one of the most popular traditional Chinese medicine (TCM) and o cially listed in Chinese Pharmacopoeia [2], and it is also a natural food additive [3]. It has been used for the treatment of nephritis, spermatorrhea, pollakiuria, asthma, and various in ammatory diseases [4].

General experimental procedures
Infrared data were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. Ultraviolet data were acquired on a Mapada UV-6100 double beam spectrophotometer. HRESIMS data were collected using a Waters Xevo G2 QTOF spectrometer. NMR spectra were recorded on a Bruker AVANCE III HD 400 NMR spectrometer. Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter. X-ray data were collected by a Rigaku Micromax-003 X-ray single-crystal diffractometer with CuKα radiation. Open column chromatography (CC) was performed by packing silica gel (200-300 mesh, Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 gel (Pharmacia Biotek, Denmark). Thin layer chromatography (TLC) was carried out on silica gel GF254 plates (Merck, Darmstadt, Germany) with 10% H 2 SO 4 in 95% ethanol followed by heating. Reversed phase semi-preparative HPLC (RP-SP-HPLC) was accomplished using an LC3000 system (Beijing Innovation Technology Co., Ltd), equipped with a phenomenon C 18 column (21.2 mm × 250 mm, 5 µm). Cells were cultured in Sanyo MCO-15 AC carbon dioxide (CO 2 ) incubator (Sanyo Electric Co., Ltd., Osaka, Japan).

Extraction and isolation
The dried mature fruits powder (47.9 kg) was extracted with 70% aqueous ethanol under re ux. After extracted for three times ( rst 479 kg for 2 h, and then 384 kg for 2 h two times), the crude extract (8.2 kg, yield 17.12%) was obtained. And then, part of the residue (6.0 kg) was suspended in H 2 O (8 L) and extracted with cHE (8 L × 8), EtOAc (8 L × 8) and n-butanol ( was separated to yield compounds 5 (11 mg, t R = 77 min), 6 (9 mg, t R = 100 min) and

X-ray Crystallographic Analysis
The X-ray crystallographic experiments were carried out on a XtaLAB Synergy R, HyPix diffractometer with CuKα radiation. Crystallographic data (No. CCDC 1993852) of 1 have been deposited at the Cambridge Crystallographic Data Center.

ECD calculations
The calculation was performed by the Gaussian 16 software. Conformation analysis were proceeded with the MMFF94s molecular mechanics force eld. Optimization of the stable conformers with a Boltzmann distribution over 1% was conducted by time-dependent density functional theory (TD-DFT) at the Cam-B3LYP/6-31 + G(d, p) level for compounds 8 and 9, with the CPCM model in MeOH. The ECD data was analysed by SpecDis v1.71 with the half-bandwidth no more than 0.3 eV. The nal ECD spectra were obtained based on the Boltzmann-calculated contribution of each conformer.
2.6. Inhibition assay on NO production RAW264.7 cells were maintained in DMEM containing 10% FBS, in a constant humidity atmosphere of 5% CO 2 and 95% air at 37°C. The cells were cultivated at a density of 3 × 10 5 cells/mL for 24 h in 96-well culture plates. And then, the cells were stimulated with LPS (1 µg/mL) and treated with various concentrations (1.5625-50 µM) of assay compounds. After exposure to the compounds for 24 h, MTT (20 µL, 5mg/mL) was added to each well [13]. Four hours later, 100 µL of lysis solution (40 g SDS, 20 mL isopropanol, 0.4 mL concentrated HCl and 400 mL ddH 2 O) was added to dissolve the formazan crystals.
The RAW264.7 cells were grown at a density of 3 × 10 5 cells/mL in 96-well culture plates. After 24 h, the cells were stimulated with LPS (1 µg/mL) and treated with various non-cytotoxic concentrations of assay compounds. And then, the cell culture supernatant (100 µL) was collected and reacted with the same volume of Griess reagent (100 µL) for 15 min at room temperature [14]. The absorbance was determined at 540 nm. The experiments were performed in parallel for three times, and L-NIL was used as a positive control. IC 50 (half maximal inhibitory concentration) value of each compound was de ned as the concentration (µM) that caused 50% inhibition of NO production. The IC 50 values were calculated by the software SPSS 16.0 (SPSS Inc., Chicago, IL, USA).

Results
Phytochemical investigation on cHE fraction of 70% ethanol extract of Psoraleae Fructus resulted in twelve unpresented bakuchiol dimmers (1-12) and ve known compounds (13-17) (Fig. 1). Structures of these new compounds were assigned by NMR spectra and single crystal X-ray diffraction. Compounds 1-3, 6-9, and 13-17 could be detected from ultrasonic extraction of Psoraleae Fructus by LC/MS analysis, suggesting that these compounds were natural products (Fig. S94). Compared with the NMR data (Tables 1 and 2) of bakuchiol [15], a side chain (3-ethenyl-3,7-dimethyl-1,6octadienyl) and a p-disubstituted benzene ring in 1 were identical to that of bakuchiol. The 1 H NMR data of an another side chain of compound 1 exhibited three methyl groups at δ H 1.78 (3H, s), 1.78 (3H, s) and presence of an α,β-unsaturated ketone group was revealed by the band at 1692 cm -1 in its IR spectrum, which was con rmed by the resonance at δ C 180.4(s) in its 13 C NMR spectrum. Comparison of the 13 C NMR spectrum of 1 with those of bakuchiol, the chemical shifts of C-3 and C-5 were shifted down eld to δ C 121.4, suggesting that this substituted group was connected to C-4 of bakuchiol moiety by an ether linkage. The full assignment of 1 H and 13 C NMR resonances was supported by 1 H-1 H COSY, DEPT, HSQC and HMBC spectral analyses. The X-ray structure was shown in Fig. 2 and con rmed the absolute con guration of 9S,9'S for 1. Thus, the structure of 1 was as shown in Fig. 1 and named bisbakuchiol M.
The plausible biosynthetic pathway of bisbakuchiol M was proposed (Fig. 3). Hydroxylation reactions occurred at the positions of C-2 and C-5 in bakuchiol to form M1. Once the 4-hydroxyl group in M1 lost a proton to generate M2-1, migrations of the double bond would start. The double bond at C-7 and C-8 would attack C-12 to form a ve-membered ring, along with the generation of carbanion at C-13 (M2-2). Subsequently, the carbanion at C-13 attacked C-6 to form six-membered ring (M2-3). The proton at C-6 left, which was accompanied by electron migrations of negative ion of oxygen to produce ketone carbonyl (M3). And then, the α-proton of double bond at C-8 was easily to be hydroxylated to generate M4. The elimination reaction would follow to the generation of M5. Similarly, the hydroxylation occurred at C-11 (M6). Subsequently, 11-hydroxyl group would be oxidized to ketone carbonyl (M7). Finally, M7 and bakuchiol were condensed to produce bisbakuchiol M. aromatic ring. Compared with the NMR data of bakuchiol, a side chain (3-ethenyl-3,7-dimethyl-1,6octadienyl) and a p-disubstituted benzene ring in 3 were identical to that of bakuchiol, together with a set of remaining NMR signals, which were very similar to those of psoracorylifol F characterized from the fruits of P. corylifolia [16]. However, the correlations between H-17' at δ H 6.43 and H-7' at δ H 2.89 were observed, which indicated that H-7' was α-oriented. The large coupling constant (J = 11.7 Hz) of H-7' and H-12' indicated a trans con guration of the two methine protons. Likewise, the con guration of H-8' was con rmed β-oriented on the basis of the large coupling constant (J = 10.6 Hz). Thus, the con guration was assigned as 7'S,8'S,9'S,12'S from the occurrence of (9S)-bakuchiol only from nature [17,18]. Furthermore, the HMBC cross-peaks of H-8' at δ H 4.06 with aromatic C-4 at δ C 159.7 correlation indicated that C-8' was connected to C-4 of bakuchiol moiety by an ether linkage (Fig. 4). According to the above data, the structure of compound 3, named bisbakuchiol O, was established as shown in Fig. 1 30 (1H, s), indicating that they were cofacial, and H-7' was assigned in a β-con guration. And the large coupling constants (J = 11.7, 10.5 Hz) indicated that H-8' and H-12' were α-oriented. As a result, the con guration was con rmed as 7'R,8'R,9'S,12'R. According to the above data, compound 4 was a dimer, whose C-8' of psoracorylifol F was connected to aromatic C-4 of bakuchiol moiety by an ether linkage (Fig. 4). Thus, the structure of compound 4, named bisbakuchiol P, was established as shown in Fig. 1. were observed. In the HMBC spectrum of 5 (Fig. 4), a psoracorylifol A unit located at C-4 of the bakuchiol unit was veri ed by correlations from H-7' at δ H 5.17 to C-4 at δ C 157. These features permitted assignment of the planar structure of compound 5 as shown in Fig. 1. In the NOESY spectrum (Fig. 5 experiments, a set of bakuchiol unit signals except for down eld shift to δ C 123.7 for C-3, C-5 and a set of psoracorylifol A unit signals except for down eld shift to δ C 82.9 for C-7' and δ C 82.0 for C-13' were observed. In the HMBC experiment (Fig. 4), a characteristic methoxyl group at δ H 3.04 (3H, s) correlated with C-7' enabled us to attach this methoxyl group to the C-7'. In NMR spectra of 6, the signals of an exomethylene of psoracorylifol A unit had disappeared, while a new signal for characteristic methyl group at δ H 0.49 (3H, s) and an oxygenated quaternary carbon at δ C 82.0 had appeared. Combined with the down eld shift of C-3 and C-5 of bakuchiol unit, it was obvious that two units were attached together by C 4 -O-C 13' . In the NOESY spectrum (Fig. 5), correlations between H-7' at δ H 4.18 and CH 3 -16'β at δ H 1.10, H-8' at δ H 3.96 and CH 3 -16'β, indicated that they were cofacial and were β-oriented. Similarly, correlations from H-8' and CH 3 -15' at δ H 0.82 supported that H-12' at δ H 3.14 was α-oriented. Finally, the structural assignment of 6 was assigned as 9S,7'S,8'S,9'S,12'S, and compound 6 was named bisbakuchiol R.
Compound 7 was isolated as white amorphous powders, and possessed the same molecular formula, C 37 H 50 O 4 , as 6 according to the HRESIMS data (m/z 557.3635 [M -H] -). Its 1D NMR pattern was highly overlapped to that of compound 6, indicating their same planar structure. In the NOESY spectrum (Fig. 5), correlations between H-7' at δ H 4.30, H-8' at δ H 3.54, and CH 3 -16'β at δ H 1.17 indicated that they were cofacial and were β-oriented. Meanwhile, correlations from H-8' and H-12' at δ H 4.08 suggested βorientation of H-12'. Finally, the structural assignment of 7 was 9S,7'S,8'S,9'S,12'R as shown in Fig. 1 and compound 7 was named bisbakuchiol.  and H-8' con rmed a trans con guration of the two methine protons of the dioxane ring [20]. Thus, the con guration of 8, named bisbakuchiol T, was established as 9S,7'S,8'S,9'S, which was supported by comparison of the calculated and experimental ECD curves ( Fig. 6).  Tables 1 and 2) of 9 were quite superimposable with those of compound 8, which clearly indicated the same skeleton as that of 8. Likely, NOE correlations between H-7' at δ H 4.91 and 16'-CH 3 at δ H 1.07, and the coupling constant (J = 6.1 Hz) between H-7' and H-8', indicated that the con guration of 9 was 9S,7'R,8'R,9'S, which was consistent with ECD data (Fig. 6). Therefore, the structure of compound 9, named bisbakuchiol U, was established as shown in Fig. 1.
Compound 10 was also isolated as yellowish oils. Its HRESIMS data exhibited a sodium adduct ion at m/z 445.3080 [M + Na] + , establishing the molecular formula as C 29 H 42 O 2 . Comparison of 1 H and 13 C NMR spectra of 10 (Table 3) and known bakuchiol, a side chain (3-ethenyl-3,7-dimethyl-1,6-octadienyl) and a p-disubstituted benzene ring of 10 were identical to that of bakuchiol. In addition, the COSY, HSQC and HMBC correlations showed the presence of 2-ethenyl-2-methyl-5-isopropanol-cyclopentan-1-ol (6ethenyl-6-methyl-4-isopropanol-cyclopentan-5-ol) substituted group in 10. Comparison of the 13 C NMR spectrum of 10 with those of bakuchiol, the chemical shifts of C-3 and C-5 in 10 were shifted down eld to δ C 124.5, suggesting that this substituted group was connected to C-4 of bakuchiol moiety by an ether linkage. The relative con guration was mainly assigned by NOESY spectrum. Furthermore, the signals of  (Table 3) of compound 10, a side chain (3-ethenyl-3,7-dimethyl-1,6-octadienyl) and a p-disubstituted benzene ring in 11 were identical to that of compound 10.
In addition, the COSY, HSQC and HMBC correlations in 11 showed the presence of clovane-2β,9α-diol [21,22] moiety with the exception of the resonances of C-1', C-2' to down eld shifts and C-3' and C-4' to high eld shifts. In the key HMBC spectrum (Fig. 4), correlations between H-2' at δ H 4.24 and C-4 at δ C 158.2 (s) revealed that C-4 of bakuchiol moiety was connected to C-2' of clovane-2β,9α-diol moiety by an ether linkage. Therefore, the structure of compound 11, named bakuchiol ether B, was de ned as shown in Fig. 1.  [22]. Comparison of the 13 C NMR spectrum of 12 with those of bakuchiol, the chemical shifts of C-3 and C-5 were shifted down eld to δ C 121.6 and the chemical shifts of C-1' was shifted down eld to δ C 80.2 in 12, suggesting that C-1' of this caryolane-1,9β-diol moiety was connected to C-4 of bakuchiol moiety by an ether linkage. Therefore, the structure of compound 12, named bakuchiol ether C, was de ned as shown in Fig. 1.
When the quaternary carbon from the other unit was connected to bakuchiol unit by C-O-C 4 , the chemical shifts of C-3 and C-5 would shift down eld (from115 to 121 or 123 ppm) as shown in compounds 1, 6, 7, 10, 12, 15, 16 and 17. Whereas, the link by CH-O-C 4 would not result in changes of δ C at C-3 and C-5 as shown in compounds 3, 4, 5 and 11. Therefore, we could infer the connection position of the dimers by the carbon chemical shifts of C-3/5 in bakuchiol unit. NO, an unstable biological free radical, comes of L-arginine under the action of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS). NO functions as a signaling molecule participating in neurotransmission and vasodilation. However, overproduction of NO is involved in in ammatory diseases, which can be treated by NO inhibitor. Compounds 1-17 (3.125-50 µM) were assay inhibition effect on NO production in LPS-stimulated RAW264.7 macrophages using the Griess reaction [14]. The MTT tests demonstrated that compound 4 showed cytotoxicity at the concentration of 50 µM, whereas other compounds were not cytotoxic. L-NIL, a selective inhibitor of iNOS, was used for the positive control.
Compound 1 exhibited signi cant inhibition of NO production with IC 50 value at the concentration of 11.47 ± 1.57 µM, which showed no signi cant difference with that of L-NIL (10.29 ± 1.10 µM).

Discussion
In our previous researches, we have obtained fourteen meroterpenoids and seventeen heterodimers of bakuchiol and evaluated their cytotoxicity [11,12]. Further investigation on the cHE extract brought about twelve unpresented bakuchiol dimmers and their NO inhibition activities were studied. In sum, Structural changes in bakuchiol increases structural and bioactive diversity of constituents from Psoraleae Fructus.
And a new skeleton compound (1) was isolated and the compound exhibited signi cant NO inhibition activities.

Conclusion
Seventeen bakuchiol dimers (1-17), including 12 undescribed dimers and 5 known compounds, were isolated from the fruits of Psoralea corylifolia L. and their structure were identi ed by spectral methods and X-ray single-crystal diffraction. Bisbakuchiol M (1), whose other bakuchiol unit was cyclized to form a 6/6/5 tricyclic system, was a new skeleton compound. And the plausible biosynthetic pathway of bisbakuchiol M was proposed. Their inhibition on NO production in LPS-stimulated RAW264.7 macrophages were evaluated by the Griess reaction. Compounds 2, 3, 10-12, 16 and 17 exhibited inhibitory activities, and the inhibition of compound 1 was equal to that of L-NIL, a positive control. These ndings suggested that Psoraleae Fructus provided natural anti-in ammatory constituents and bisbakuchiol M had the potential to be novel NO inhibitor.